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Received: 28 April 2019    Revised: 30 June 2019    Accepted: 8 July 2019

DOI: 10.1111/jfbc.12996

FULL ARTICLE

Antityrosinase mechanism of ellagic acid in vitro and its effect

on mouse melanoma cells

Qian Huang | Wei‐Ming Chai  | Zuo‐Yuan Ma | Wei‐Liang Deng | Qi‐Ming Wei |

Shuang Song | Zheng‐Rong Zou | Yi‐Yuan Peng

College of Life Science, and Key Laboratory

of Functional Small Organic Molecule,
Ministry of Education, Jiangxi Normal
University, Nanchang, China
Correspondence
Wei‐Ming Chai, College of Life Science, and
Key Laboratory of Functional Small Organic
Molecule, Ministry of Education, Jiangxi
Normal University, Nanchang, 330022,
China.
Email: chaiweiming@jxnu.edu.cn
Abstract
The activities of ellagic acid in inhibiting mushroom tyrosinase and cell proliferation
were evaluated in this research. The results of enzyme kinetics indicated that ellagic
acid could effectively inhibit tyrosinase activity. The value of the semi‐inhibitory rate
(IC50) was 0.2 ± 0.05 mM. Ellagic acid inhibited tyrosinase activity in a reversible
manner and was a mixed tyrosinase inhibitor. Furthermore, ellagic acid had a good
inhibitory effect on the proliferation of mouse melanoma B16 cells and could induce
apoptosis. The results acquired from fluorescence spectroscopy revealed that the
interaction of ellagic acid with tyrosinase depended on hydrogen bond and electro‐
static force. In addition, computational docking showed that ellagic acid interacted
with amino acid residues of tyrosinase (Asn19 and Lys372) by hydrogen bond and
produced electrostatic interaction with amino residue Lys18.
Practical applications
In the present research, the antityrosinase mechanism of ellagic acid and its effect
on mouse melanoma cells were investigated. This study suggested that ellagic acid
had a strong inhibitory activity against tyrosinase and cell proliferation,which laid
an experimental foundation for the development of new drugs and whitening prod‐
ucts. The combined multispectral methods used in this research can be applied to the
screening of other antityrosinase inhibitors, further promoting the development and
utilization of tyrosinase inhibitors.

KEYWORDS
ellagic acid, fluorescence quenching, inhibitory mechanism, molecular docking, mouse
melanoma cells, tyrosinase

1 |  I NTRO D U C TI O N
Tyrosinase is a class of multifunctional oxidoreductases widely found
in animals, plants, and microorganisms. Tyrosinase is the key enzyme
that catalyzes tyrosine to form melanin (Orhan & Khan, 2014; Zhuang
et al., 2010). It has two catalytic activities. Its monophenolase activity
hydroxylates L‐tyrosine to L‐dopa. Then, its diphenolase activity oxi‐
dizes L‐dopa to dopaquinone, and finally forms melanin after a series
of reactions (Cui et al., 2015; Kanteev, Goldfeder, & Fishman, 2015;
Liu et al., 2013). However, excessive melanin can lead to pigmentation
diseases, for example, age‐related skin hyperpigmentation, chloas‐
mas, and freckles (Huang et al., 2006; Wang, Chai, Yang, Wei, & Peng,
2016). Tyrosinase is also a key enzyme in controlling the nutritive value
and quality of plant‐derived foods (Kim & Uyama, 2005). Hence, ef‐
ficient and secure inhibitor of tyrosinase is crucial in preventing the
skin diseases in humans and improving food quality (Chai et al., 2012).
Ellagic acid (Figure 1a) is a natural polyphenol component generally
distributed in various nuts, soft fruits, and other plant tissues (Pitchakarn

J Food Biochem. 2019;00:e12996. wileyonlinelibrary.com/journal/jfbc © 2019 Wiley Periodicals, Inc.  |  1 of 9

https://doi.org/10.1111/jfbc.12996
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(a) O (c)

HO
O

OH
HO

O
OH

(b) (d)
(d1)

(d2)

F I G U R E 1   Chemical structure of ellagic acid (a); Inhibitory effect of ellagic acid on the enzyme activity of tyosinase (b); inhibitory
mechanism (c), concentrations of ellagic acid for curves 1–5 were 0, 300, 400, 500, 600 µM, respectively; inhibitory type and inhibition
constants (d), concentrations of ellagic acid for curves 1–4 were 0, 300, 400, 600 µM, respectively. The plot of slope versus the
concentrations of the ellagic acid for determining the inhibition constant KI (d1). The plot of intercept versus the concentrations of the ellagic
acid for determining the inhibition constant KIS (d2)

et al., 2013). In recent years, the anticancer and antimutagenic effects 2.2 | Enzymatic kinetics experiments
of ellagic acid have attracted extensive attention of researchers (Wang
et al., 2012; Zhang et al., 2014). In addition, ellagic acid also possesses In this experiment, the method of measuring activity of enzyme

antioxidant activity (Ceribasi, Sakin, Turk, Sonmez, & Atessahin, 2012; was based on the method of Chai et al. (2018). The concentration of

Turk, Ceribasi, Sakin, Sonmez, & Atessahin, 2010). However, there are L‐dopa was 5 mmol/L, and the temperature of the experiment was

no reports about the antityrosinase activity of ellagic acid and its mech‐ 30°C. In 3‐ml system, 0.3 ml of substrate, 0.75 ml of buffer solu‐

anism. Hence, the antityrosinase activity of ellagic acid was studied in tion, distilled water 1.8 ml, and 0.1 ml of ellagic acid (100, 200, 300,

this research and its inhibition mechanism was elucidated by enzymatic 400, 500, 600 µM) were added in turn. Finally, 0.05 ml of mushroom

experiments, fluorescence spectroscopy and computer molecular sim‐ tyrosinase solution was added and shaken immediately. The curves

ulation. Moreover, we studied the anti‐proliferation of ellagic acid on of absorbance varying with time (wavelength at 475 nm) were meas‐

mouse melanoma cells. The aim of this research was to find a new ty‐ ured by Beckmann DU‐730 UV–Vis spectrophotometer (USA). The

rosinase inhibitor and elucidate its inhibition mechanism, so as to pro‐ slope of the resulting line was the enzyme activity (ΔOD/Δt). Three

vide a scientific basis for its development and utilization. parallel experiments were performed on the enzyme activity assay.
The inhibitory mechanism was determined by altering the con‐
centration of tyrosinase and the method was the same as the de‐
2 |   E X PE R I M E NTA L termination of tyrosinase activity. The type of inhibition can be
determined via Lineweaver–Burk plot, and inhibition constants were
2.1 | Reagents and equipment determined from the secondary plot (Chai, Lin, Wang, et al., 2017).

Ellagic acid, human serum albumin (HSA), and thiazolyl blue tetra‐
zolium bromide (MTT) were products of Aladdin Industrial Co.
(Shanghai, China). Tyrosinase (EC 1.14.18.1) is derived from mush‐
room and its specific activity was 1,000 U/mg. Tyrosinase and its
substrate (L‐dopa) were purchased from Sigma‐Aldrich (St. Louis,
MO, USA). Ethidium bromide (EB) and acridine orange (AO) were
products of Amresco (Solon, OH, USA).
2.3 | Cell culture and MTT assay
Under sterile conditions, B16 melanoma cells were inoculated into
medium containing 10% fetal bovine serum (FBS), 100 U/mL penicil‐
lium, and streptomycin sulfate 0.1 g/L. After incubation in 5% CO2
cell incubator (at 37°C, 24 hr), on the third day of inoculation, 0.25%
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trypsin was used to digest the cells. The cells used in the experiment If there are slightly changes about enthalpy under different tem‐
were in logarithmic growth phase. MTT assay was used to detect cell peratures, it can be considered constant. Subsequently, through the
proliferation and activity (Chai, Lin, Feng, Zou, & Wang, 2017). van't Hoff equation, we can obtain the values of entropy changes
(ΔS°), enthalpy changes (ΔH°), and free energy changes (ΔG°) (Xu
et al., 2013).
2.4 | AO/EB fluorescent staining
ΔH◦ ΔS◦
The cells were digested with 0.25% trypsin and then mixed into log KA = − + (3)
2.303RT 2.303R
suspension. After incubation for 24 hr, the cells were mixed with
100 µg/ml AO and 100 µg/ml ethidine olfactoride (EB). DP‐50 fluo‐ ΔG◦ = ΔH◦ TΔS◦ (4)
rescent microscope (Olympus, Tokyo, Japan) was used to observe
the morphology of the cells (Liu, Liu, Liu, & Wu, 2015). In the formula, the value of R is 8.314 J mol−1 K−1, which is the
gas constant.
Compared with fluorescence spectroscopy, three‐dimensional
2.5 | Fluorescence quenching assay fluorescence spectroscopy can obtain not only excitation wave‐

Fluorescence spectrophotometer was employed to study the fluo‐ length and emission wavelength, but also fluorescence intensity in‐

rescence quenching assay. Based on previous report (Lin et al., formation when changing. The excitation and emission wavelength

2018), we have carried out fluorescence quenching experiments. ranged from 200 to 600 nm in the experiment (Ding, Hu, Xu, Zhang,

The original solution concentration of ellagic acid was 3 mM and dis‐ & Gong, 2018). The measurement method was similar to that of flu‐

solved in dimethyl sulfoxide. The concentrations of ellagic acid were orescence spectroscopy.

set to 5, 10, 15, 20, 25, and 30 µM, respectively. The volume ratio
of sodium phosphate buffer, tyrosinase, and ellagic acid was 8:1:1.
After mixing the three solutions evenly, they were cultured at 30°C
for 30 s and then measured in a fluorescent cuvette. Measurement
parameter settings were based on reference (Chai et al., 2014).
2.6 | Determination of energy transfer between
tyrosine and ellagic acid
In accordance with Förster's theory of nonradiative energy transfer

The Stern–Volmer equation was listed below (Eftink & Ghiron, (Liu, Qi, & Li, 2010), we can calculate the binding distance between

1981). It was used to calculate some parameters related to fluores‐ ellagic acid and tyrosinase. The formula is as follows.

cence quenching.
R60 F0 − F
E= = (5)
R60 + r6 F0
F0
= 1 + Kq 𝜏0 [Q] = 1 + KSV [Q] (1)
F −4 2
R60 = 8.79 × 10−25 N K J𝜙 (6)

Among the formula, F0 and F indicate the fluorescence inten‐ ∑

F(𝜆) 𝜀(𝜆) 𝜆4 Δ𝜆
sity of tyrosinase in the absence and presence of ellagic acid. The
J= ∑ (7)
F(𝜆) Δ𝜆
ellagic acid concentration is represented by [Q]. Ksv means the Stern–
Volmer quenching constant. Kq is the biomolecular quenching rate Fx Ast
𝜙 = 𝜙st (8)
constant. τ 0, whose value is 10−8 s, represents the lifetime of fluo‐ Fst Ax
rescent group without quenching agent.
Static and dynamic are usual mechanisms for explaining fluo‐ In these equations, when the transmission efficiency is 50%, R0 is

rescence quenching (Li et al., 2005). In static quenching interaction, critical distance. F0 and F are the same as in Equation 1. K 2 is dipole

while ellagic acid is freely bound to a set of equivalent sites of the spatial orientation factor with a value of 2/3. J is the overlapping

enzyme, the binding constant (K A) and number of binding sites (n) integral of donor fluorescence spectrum and receptor ultraviolet–

can be calculated (Shahabadi, Maghsudi, & Rouhani, 2012).
(2)
[( ) ] [ ]
log F0 − F ∕F = n log Q + log KA
Four main types of noncovalent interactions: electrostatic inter‐
action, van der Waals interaction, hydrogen bond, and hydrophobic
force occurred in ligand–macromolecule binding (Zeng, Zhang, Liao,
& Gong, 2016). To further reveal the interactions between tyrosi‐
nase and ellagic acid, thermodynamic parameters were calculated by
changing temperature (293, 303, and 310 K).
visible absorption spectrum. The value of N is 1.336, which rep‐
resents the refractive index of the medium. Ɛ(λ) denotes the molar
absorption coefficient of acceptor.
The value of φst is 0.13, which represents fluorescence quantum
yields of HSA.
2.7 | Molecular docking
A crystal structure of tyrosinase PPO3 from Agaricus bisporus (pdb
entry 2Y9W) was selected as source of the X‐ray structure of tyrosi‐
nase. (Chai, Lin, Song, et al., 2017). The three‐dimensional structure
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of ellagic acid was prepared using Chem Bio Draw Ultra 8.0. After
preparing the molecular structure, the molecular simulation was
docked using an Autodock tools. The lowest free energy conforma‐
tion is used as the final result of molecular docking (Anantharaman,
Hemachandran, Priya, Sankari, & Siva, 2015).

3 |  R E S U LT S A N D D I S CU S S I O N

3.1 | Inhibition effect
As can be seen from the Figure 1b, ellagic acid could effectively in‐
hibit tyrosinase activity. Compared with arbutin (Thanigaimalai et al.,
2010) (IC50 = 180 µM), amoxicillin (Chen et al., 2013) (IC50 = 900 µM),
and hesperetin (Si et al., 2012) (IC50 = 11.25 ± 1.73 mM), the value of
semi‐inhibitory rate of ellagic acid was 0.2 ± 0.05 mM. These results
of activity assay showed that ellagic acid was an effective tyrosinase
inhibitor.

3.2 | Inhibition mechanism
Different concentrations of ellagic acid (0, 300, 400, 500, 600 µM)
were selected to inhibit tyrosinase to obtain relationship between
residual enzyme activity and enzyme concentrations. As can be seen
F I G U R E 2   Effect of ellagic acid on cell proliferation (a), the
from the Figure 1c, all straight lines with different concentrations of
concentrations of the ellagic acid was 0.125, 0.25, 0.5, and 1.0 µM,
ellagic acid passed through the origin. These results revealed ellagic respectively; AO/EB double‐staining of B16 mouse melanoma cells
acid was a reversible inhibitor of enzyme. in the presence of ellagic acid with different concentrations. The
concentrations for the b1, b2, b3, and b4 were 0, 0.25, 0.5, and
1 µM, respectively. Decreased number of living cells and apoptosis
3.3 | Inhibition type and constants were observed at 200× magnification

Through Lineweaver–Burk plot, we can determine the type of in‐

hibition of tyrosinase by ellagic acid. In Figure 1d, the different
lines intersected in the second quadrant, revealing that ellagic acid
was a mixed inhibitor. From Figure 1d1 and d2, we can calculate
that the inhibition constants KI was 0.14 ± 0.02 mM and KIS was
0.91 ± 0.05 mM. The KI value of the inhibitor was lower than the KIS
value, indicating that the inhibitor has a stronger affinity to enzyme
than enzyme–substrate complex.
3.4 | Cytological effect of ellagic acid on B16 mouse
melanoma cells
As shown in Figure 2a, with increase concentrations of ellagic acid, the
number of cells decreased and the mortality increased. In addition, the
cell mortality rate increased to 92 ± 0.8% when the concentration of
ellagic acid reached 1.0 µM. These results indicated that ellagic acid
had a strong inhibitory effect on cell proliferation.
Dual AO/EB fluorescent staining was employed to distinguish liv‐
ing cells and apoptotic cells. AO can penetrate intact cells and stain
the nucleus green, whereas EB can only penetrate the cells with
damaged membrane and stain the nucleus red (Kumar et al., 2018).
As can be seen from the Figure 2b1, the control group cells showed
bright green fluorescence and complete cell structure. The cells
treated with ellagic acid partly showed orange‐red fluorescence. The
orange‐red fluorescent cells gradually appeared and increased with
the concentration of ellagic acid increasing (Figure 2b2–b4). These
results showed ellagic acid induced cell apoptosis of B16 melanoma
cells.
3.5 | Fluorescence quenching
After excitation at 290 nm, the fluorescence intensity of tyrosi‐
nase reached a peak at 341 nm (Figure 3a, curve 1). In Figure 3a,
curves 2–7 represented the concentration of ellagic acid increased
gradually. With the addition of ellagic acid to tyrosinase solu‐
tion, the fluorescence intensity of the enzyme reduced progres‐
sively, suggesting that ellagic acid may interact with tyrosinase.
In Figure 3b, when relative fluorescence intensity of enzyme
was lowered to 50%, the ellagic acid concentration was 15 μM.
Furthermore, the blue shift can be detected in Figure 3a, which
revealed that ellagic acid can lead to changes in the conformation
of tyrosinase.
In Figure 3c and Table 1, based on Stern–Volmer equation, KSV
and Kq of ellagic acid on tyrosinase were calculated. The conse‐
quences showed that when the temperature rose, the KSV values
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F I G U R E 3   Fluorescence spectra of tyrosinase in the presence of ellagic acid at different concentrations (a), the concentrations of
ellagic acid for curves 1–7 were 0, 5, 10, 15, 20, 25, and 30 µM, respectively. Curve a was fluorescence intensity of ellagic acid at the
concentrations of 30 µM. The relative intensity of tyrosinase at different concentrations of ellagic acid (b). The Stem–Volmer plots for the
fluorescence quenching of tyrosinase by ellagic acid at different temperatures (c), the temperatures for curves 1–3 were 293, 303, and
310 K, respectively. Plots of log [(F0 − F)/F] against log [Q] for tyrosinase and various concentrations of ellagic acid at different temperatures
(d), the temperatures for curves 1–3 were 293, 303, and 310 K, respectively. F0 and F was the fluorescence intensities of tyrosinase in
the absence and presence of ellagic acid, [Q] represented the concentration of ellagic acid. The spectral overlaps (e) of the fluorescence
spectrum of tyrosinase (a) with the absorption spectrum of ellagic acid (b)

gradually decreased, indicating that static quenching occurred due
to the formation of an ellagic acid–enzyme complex.
It can be calculated that Kq were 1.23 × 1013, 1.05 × 1013,
and 1.02 × 1013 L mol−1 S−1 at 293, 303, and 310 K, respectively.
Compared with the maximum scattering collision quenching con‐
stants of biological molecules (2.0 × 1010 L mol−1 S−1), the values of
these Kq were three orders of magnitude larger than it (Miao, Jiang,
Jiang, Zhang, & Li, 2015; Xiao, Gu, Liang, Li, & Luo, 2014). Therefore,
the quenching process was static process that formed complex,
instead of dynamic process caused by molecular collision.
For the static quenching, K A and n could be calculated by
log[(F0 − F)/F] against log[Q] (Figure 3d), and the results were shown
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6 of 9       HUANG et al.

TA B L E 1   The quenching constants KSV and binding constants K A , the number of binding sites n, and relative thermodynamic parameters
of the interaction between ellagic acid and tyrosinase at different temperatures

T (K) Ksv (10 4 L/mol) Ra K A (10 4 L/mol) n Rb △Ho(kJ/mol) △Go(kJ/mol) △So(J mol−1 K−1)

293 12.31 ± 0.3 0.9471 10.29 ± 0.2 1.02 ± 0.04 0.9459 −51.29 ± 1.3 −74.69 ± 1.4 79.86 ± 1.6

303 10.54 ± 0.5 0.9345 3.72 ± 0.4 1.25 ± 0.02 0.9367 −75.48 ± 1.2
310 10.18 ± 0.6 0.9721 3.40 ± 0.6 1.28 ± 0.04 0.9345 −76.05 ± 1.0
a b
Note: R is the correlation coefficient for the KSV values. R is the correlation coefficient for the K A values.

F I G U R E 4   Three‐dimensional fluorescence spectra of tyrosinase (a) and tyrosinase–ellagic acid complex (b). (a):C(tyrosinase) = 0.2 mg/ml;
(b):C(tyrosinase) = 0.2 mg/ml, C(ellagic acid)= 30 µM

in Table 1. As temperature increased, the binding constant gradually Calculating thermodynamic parameters by van't Hoff equation
decreased, which suggested that the capacity of ellagic acid binding can further characterize the intermolecular interaction between ty‐
to tyrosinase became weaker at higher temperature. rosinase and ellagic acid. As shown in Table 1, it could be seen that
HUANG et al. |
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TA B L E 2   Three‐dimensional fluorescence spectral characteristics of tyrosinase and tyrosinase–ellagic acid complex

Tyrosinase Tyrosinase–ellagic acid complex

Peak position Stokes Peak position Stokes

Fluorescence peaks λex/λem (nm/nm) Δλ (nm) Intensity F λex/λem (nm/nm) Δλ (nm) Intensity F

peak 1 200/200 → 600/600 0 153.2 → 6,028.0 200/200 → 600/600 0 94.1 → 3,971.0

peak 2 280/340 60 286.1 280/340 50 230.8
peak 3 260/520 260 1,098.0 260/520 260 551.6

F I G U R E 5   Molecular docking results of ellagic acid on tyrosinase: the secondary structure of tyrosinase–ellagic acid complex (a); the
tertiary structure of tyrosinase–ellagic acid complex (b); hydrogen bond between ellagic acid and amino residues (c); electrostatic interaction
of ellagic acid with amino residues (d)

ΔH° = −51.29 ± 1.3 kJ/mol, which showed the process of complexing
ellagic acid with enzyme was exothermic reaction. Meanwhile, the
result of ΔG° < 0, suggested that the interaction between tyrosinase
and ellagic acid occurred spontaneously (Chung, Rojanasasithara,
Mutilangi, & McClements, 2016). The result of ΔH° < 0, suggested
there was hydrogen bond between enzyme and ellagic acid.
Additionally, the results of ΔH° < 0, ΔS° > 0 manifested there was
electrostatic force between enzyme and ellagic acid.
3.6 | Energy transfer between tyrosinase and
ellagic acid
In Figure 3e, the gray part represented the overlapping part of fluo‐
rescence spectrum of enzyme and ultraviolet spectrum of ellagic
acid. Based on Equation 8, the φ value was computed to be 0.24.
From Equations 5–7, J was computed to be 3.77 × 10–12 cm3 L mol−1.
The value of R0 was 7.47 nm, E was 0.63 and r was 6.8 nm. The value
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of r was less than 8 nm and 0.5R0 < r < 1.5R0, suggesting that there
was nonradiative energy transfer from enzyme to ellagic acid (Wang,
Zhang, Yan, & Gong, 2014).
3.7 | Three‐dimensional fluorescence spectroscopy
In Figure 4 and Table 2, peak 1 and peak 2 denote the Rayleigh scatter‐
ing peak (λex = λem) (Zhang, Dai, Zhang, Yang, & Liu, 2008) and spectral
characteristic of tryptophan and tyrosine residues (λex = 280.0 nm,
λex = 340.0 nm), respectively. Peak 3 (λex = 260.0 nm, λex = 520.0 nm)
mainly revealed the fluorescence of the polypeptide backbone struc‐
ture of tyrosinase as a result of n  →  π* transition. After adding el‐
lagic acid to tyrosinase solution, the fluorescence intensity of peak 2
decreased from 286.1 to 230.8, and that of peak 3 decreased from
1,098.0 to 551.6. These results indicated the conformational change
of the tyrosinase occurred when the ellagic acid binding to it.
3.8 | Computational docking
Computational docking was employed to further understand in‐
hibitory mechanism of ellagic acid on the tyrosinase (Azam, Uddin,
Syed, & Ul‐Haq, 2012). Figure 5a,b offered the binding mode and
surface of binding site. The interaction between ellagic acid and
tyrosinase were mainly driven by hydrogen bond and electrostatic
forces. In Figure 5c, the ellagic acid formed hydrogen bonds with
the two amino residues (Asn19 and Lys372). The electrostatic in‐
teraction was generated by the amino residues Lys18 (Figure 5d).
These results showed that, consistent with the thermodynamic
analysis results, the intermolecular forces of ellagic acid and ty‐
rosinase were hydrogen bonds and electrostatic forces.
4 |  CO N C LU S I O N
In conclusion, the principal findings of this study concluded below: (i)
Ellagic acid was an efficient, reversible, and competitive–noncompeti‐
tive mixed‐type inhibitor of tyrosinase. (ii) The reason that ellagic acid
quenched the fluorescence of tyrosinase was the formation of ellagic
acid–tyrosinase complex in a static mechanism with the nonradiation
energy transfer. (iii) Hydrogen bonds and electrostatic forces were
the intermolecular forces of ellagic acid and enzyme. (iv) Ellagic acid
changed the conformation of tyrosinase. (v) Ellagic acid had a good
inhibitory effect on the proliferation of B16 mouse melanoma cells and
could induce apoptosis. Therefore, our study demonstrated that el‐
lagic acid was an alternative tyrosinase inhibitor, which offered a sci‐
entific basis for its possible application for treating skin diseases.
AC K N OW L E D G M E N T S
Sources of funding for this study: Jiangxi Natural Science Foundation
(No. 20171BAB214019), Jiangxi Education Department Project
(No. GJJ150302), and Graduate Innovation Fund Project of Jiangxi
Normal University (No. YJS2018079).
C O N FL I C T O F I N T E R E S T
We have no conflicts of interest to declare.
ORCID
Wei‐Ming Chai  https://orcid.org/0000-0001-9163-8138
REFERENCES
Anantharaman, A., Hemachandran, H., Priya, R. R., Sankari, M., & Siva, R.
(2015). Inhibitory effect of apocarotenoids on the activity of tyrosi‐
nase: Multi‐spectroscopic and docking studies. Journal of Bioscience
and Bioengineering, 121, 13–20.
Azam, S. S., Uddin, R., Syed, A. A. S., & Ul‐Haq, Z. (2012). Molecular
docking studies of potent inhibitors of tyrosinase and α‐glucosi‐
dase. Medicinal Chemistry Research, 21, 1677–1683. https​ ://doi.
org/10.1007/s00044-011-9684-3
Ceribasi, A. O., Sakin, F., Turk, G., Sonmez, M., & Atessahin, A. (2012).
Impact of ellagic acid on adriamycin‐induced testicular histopatho‐
logical lesions, apoptosis, lipid peroxidation and sperm damages.
Experimental and Toxicologic Pathology, 64, 717–724. https​ ://doi.
org/10.1016/j.etp.2011.01.006
Chai, W. M., Huang, Q., Lin, M. Z., Ou‐Yang, C., Huang, W. Y., Wang,
Y. X., … Feng, H. L. (2018). Condensed tannins from longan bark
as inhibitor of tyrosinase: Structure, activity, and mechanism.
Journal of Agricultural and Food Chemistry, 66, 908–917. https​://doi.
org/10.1021/acs.jafc.7b05481
Chai, W. M., Lin, M. Z., Feng, H. L., Zou, Z. R., & Wang, Y. X. (2017).
Proanthocyanidins purified from fruit pericarp of Clausena lansium
(Lour.) Skeels as efficient tyrosinase inhibitors: Structure evaluation,
inhibitory activity and molecular mechanism. Food & Function, 8,
1043–1051. https​://doi.org/10.1039/C6FO0​1320A​
Chai, W. M., Lin, M. Z., Song, F. J., Wang, Y. X., Xu, K. L., Huang, J. X.,
… Peng, Y. Y. (2017). Rifampicin as a novel tyrosinase inhibitor:
Inhibitory activity and mechanism. International Journal of Biological
Macromolecules, 102, 425–430. https​ ://doi.org/10.1016/j.ijbio​
mac.2017.04.058
Chai, W. M., Lin, M. Z., Wang, Y. X., Xu, K. L., Huang, W. Y., Pan, D. D.,
… Peng, Y. Y. (2017). Inhibition of tyrosinase by cherimoya pericarp
proanthocyanidins: Structural characterization, inhibitory activity
and mechanism. Food Research International, 100, 731–739. https​://
doi.org/10.1016/j.foodr​es.2017.07.082
Chai, W. M., Shi, Y., Feng, H. L., Qiu, L., Zhou, H. C., Deng, Z. W., …
Chen, Q. X. (2012). NMR, HPLC‐ESI‐MS, and MALDI‐TOF MS anal‐
ysis of condensed tannins from Delonix regia (Bojer ex Hook.) Rat
and their bioactivities. Journal of Agricultural and Food Chemistry, 60,
5013–5022.
Chai, W. M., Shi, Y., Feng, H. L., Xu, L., Xiang, Z. H., Gao, Y. S., & Chen, Q.
X. (2014). Structure characterization and anti‐tyrosinase mechanism
of polymeric proanthocyanidins fractionated from kiwifruit pericarp.
Journal of Agricultural and Food Chemistry, 62, 6382–6389. https​://
doi.org/10.1021/jf501​0 09v
Chen, X. X., Zhang, J., Chai, W. M., Feng, H. L., Xiang, Z. H., Shen, D.
Y., & Chen, Q. X. (2013). Reversible and competitive inhibitory
­kinetics of amoxicillin on mushroom tyrosinase. International Journal
of Biological Macromolecules, 62, 726–733. https​://doi.org/10.1016/
j.ijbio​mac.2013.09.052
Chung, C., Rojanasasithara, T., Mutilangi, W., & McClements, D. J.
(2016). Stabilization of natural colors and nutraceuticals: Inhibition
of anthocyanin degradation in model beverages using polyphenols.
Food Chemistry, 212, 596–603. https​ ://doi.org/10.1016/j.foodc​
hem.2016.06.025
HUANG et al.
Cui, Y., Liang, G., Hu, Y. H., Shi, Y., Cai, Y. X., Gao, H. J., … Wang, Q.
(2015). Alpha‐substituted derivatives of cinnamaldehyde as ty‐
rosinase inhibitors: Inhibitory mechanism and molecular analysis.
Journal of Agricultural and Food Chemistry, 63, 716–722. https​://doi.
org/10.1021/jf505​469k
Ding, H., Hu, X., Xu, X., Zhang, G., & Gong, D. (2018). Inhibitory mech‐
anism of two allosteric inhibitors, oleanolic acid and ursolic acid on
α‐glucosidase. International Journal of Biological Macromolecules, 107,
1844–1855. https​://doi.org/10.1016/j.ijbio​mac.2017.10.040
Eftink, M. R., & Ghiron, C. A. (1981). Fluorescence quenching studies
with proteins. Analytical Biochemistry, 114, 199–227. https​ ://doi.
org/10.1016/0003-2697(81)90474-7
Huang, X. H., Chen, Q. X., Wang, Q., Song, K. K., Wang, J., Sha, L., &
Guan, X. (2006). Inhibition of the activity of mushroom tyrosinase by
alkylbenzoic acids. Food Chemistry, 94, 1–6. https​://doi.org/10.1016/
j.foodc​hem.2004.09.008
Kanteev, M., Goldfeder, M., & Fishman, A. (2015). Structure‐function
correlations in tyrosinases. Protein Science, 24, 1360–1369. https​://
doi.org/10.1002/pro.2734
Kim, Y. J., & Uyama, H. (2005). Tyrosinase inhibitors from natural and
synthetic sources: Structure, inhibition mechanism and perspective
for the future. Cellular and Molecular Life Sciences, 62, 1707–1723.
https​://doi.org/10.1007/s00018-005-5054-y
Kumar, N. P., Thatikonda, S., Tokala, R., Kumari, S. S., Lakshmi, U. J.,
Godugu, C., … Kamal, A. (2018). Sulfamic acid promoted one‐pot
synthesis of phenanthrene fused‐dihydrodibenzo‐quinolinones:
Anticancer activity, tubulin polymerization inhibition and apoptosis
inducing studies. Bioorganic Medicinal Chemistry, 26, 1996–2008.
https​://doi.org/10.1016/j.bmc.2018.02.050
Li, Y., He, W., Liu, J., Sheng, F., Hu, Z., & Chen, X. (2005). Binding of
the bioactive component jatrorrhizine to human serum albumin.
Biochimica et Biophysica Acta (BBA)—General Subjects, 1722, 15–21.
https​://doi.org/10.1016/j.bbagen.2004.11.006
Lin, M. Z., Chai, W. M., Ou‐Yang, C., Huang, Q., Xu, X. H., & Peng, Y. Y.
(2018). Antityrosinase mechanism of omeprazole and its application
on the preservation of fresh‐cut Fuji apple. International Journal of
Biological Macromolecules, 117, 538–545. https​ ://doi.org/10.1016/
j.ijbio​mac.2018.05.172
Liu, E. H., Qi, L. W., & Li, P. (2010). Structural relationship and binding
mechanisms of five flavonoids with bovine serum albumin. Molecules,
15, 9092–9103. https​://doi.org/10.3390/molec​ules1​5129092
Liu, K., Liu, P. C., Liu, R., & Wu, X. (2015). Dual AO/EB staining to de‐
tect apoptosis in osteosarcoma cells compared with flow cytometry.
Medical Science Monitor Basic Research, 21, 15–20.
Liu, X. X., Sun, S. Q., Wang, Y. J., Xu, W., Wang, Y. F., Park, D., … Han,
H. Y. (2013). Kinetics and computational docking studies on the in‐
hibition of tyrosinase induced by oxymatrine. Applied Biochemistry
and Biotechnology, 169, 145–158. https​ ://doi.org/10.1007/
s12010-012-9960-9
Miao, M., Jiang, B., Jiang, H., Zhang, T., & Li, X. (2015). Interaction
mechanism between green tea extract and human α‐amylase for
reducing starch digestion. Food Chemistry, 186, 20–25. https​://doi.
org/10.1016/j.foodc​hem.2015.02.049
Orhan, I. E., & Khan, M. T. H. (2014). Flavonoid derivatives as potent
tyrosinase inhibitors—A survey of recent findings between 2008–
2013. Current Topics in Medicinal Chemistry, 14, 1486–1493.
Pitchakarn, P., Chewonarin, T., Ogawa, K., Suzuki, S., Asamoto, M.,
Takahashi, S., … Limtrakul, P. (2013). Ellagic acid inhibits migra‐
tion and invasion by prostate cancer cell lines. Asian Pacific Journal
of Cancer Prevention, 14, 2859–2863. https​ ://doi.org/10.7314/
APJCP.2013.14.5.2859
Shahabadi, N., Maghsudi, M., & Rouhani, S. (2012). Study on the in‐
teraction of food colourant quinoline yellow with bovine serum
|
      9 of 9
albumin by spectroscopic techniques. Food Chemistry, 135, 1836–1841.
https​://doi.org/10.1016/j.foodc​hem.2012.06.095
Si, Y. X., Wang, Z. J., Park, D., Chung, H. Y., Wang, S. F., Yan, L., … Park,
Y. D. (2012). Effect of hesperetin on tyrosinase: Inhibition kinetics
integrated computational simulation study. International Journal of
Biological Macromolecules, 50, 257–262. https​ ://doi.org/10.1016/
j.ijbio​mac.2011.11.001
Thanigaimalai, P., Sharma, V. K., Lee, K. C., Yun, C. Y., Kim, Y., & Jung,
S. H. (2010). Refinement of the pharmacophore of 3,4‐dihydro‐
quinazoline‐2(1H)‐thiones for their anti‐melanogenesis activity.
Bioorganic & Medicinal Chemistry Letters, 20, 4771–4773. https​://doi.
org/10.1016/j.bmcl.2010.06.123
Turk, G., Ceribasi, A. O., Sakin, F., Sonmez, M., & Atessahin, A. (2010).
Antiperoxidative and anti‐apoptotic effects of lycopene and ellagic
acid on cyclophosphamide‐induced testicular lipid peroxidation
and apoptosis. Reproduction Fertility and Development, 22, 587–596.
https​://doi.org/10.1071/RD09078
Wang, N., Wang, Z.‐Y., Mo, S.‐L., Loo, T. Y., Wang, D.‐M., Luo, H.‐B., …
Chen, J.‐P. (2012). Ellagic acid, a phenolic compound, exerts anti‐
angiogenesis effects via vegfr‐2 signaling pathway in breast cancer.
Breast Cancer Research and Treatment, 134, 943–955. https​ ://doi.
org/10.1007/s10549-012-1977-9
Wang, R., Chai, W. M., Yang, Q., Wei, M. K., & Peng, Y. Y. (2016). 2‐(4‐
fluorophenyl)‐quinazolin‐4(3H)‐one as a novel tyrosinase inhibitor:
Synthesis, inhibitory activity, and mechanism. Bioorganic & Medicinal
Chemistry Letters, 24, 4620–4625. https​ ://doi.org/10.1016/
j.bmc.2016.07.068
Wang, Y. J., Zhang, G. W., Yan, J. K., & Gong, D. M. (2014). Inhibitory
effect of morin on tyrosinase: Insights from spectroscopic and mo‐
lecular docking studies. Food Chemistry, 163, 226–233. https​://doi.
org/10.1016/j.foodc​hem.2014.04.106
Xiao, F., Gu, M., Liang, Y., Li, L., & Luo, Y. (2014). Spectroscopic investi‐
gation on the interaction of hyperbranched poly (amine) ester with
model plasma protein: Effect on the structural and conformational
changes. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, 118, 1106–1112.
Xu, H., Yao, N., Xu, H., Wang, T., Li, G., & Li, Z. (2013). Characterization
of the interaction between eupatorin and bovine serum albumin by
spectroscopic and molecular modeling methods. International Journal
of Molecular Sciences, 14, 14185–14203. https​ ://doi.org/10.3390/
ijms1​4 0714185
Zeng, L., Zhang, G., Liao, Y., & Gong, D. (2016). Inhibitory mechanism
of morin on α‐glucosidase and its anti‐glycation properties. Food &
Function, 7, 3953–3963. https​://doi.org/10.1039/C6FO0 ​0680A​
Zhang, H. M., Zhao, L., Li, H., Xu, H., Chen, W. W., & Tao, L. (2014).
Research progress on the anticarcinogenic actions and mechanisms
of ellagic acid. Cancer Biology and Medicine, 11, 92–100.
Zhang, Y. Z., Dai, J., Zhang, X. P., Yang, X., & Liu, Y. (2008). Studies of the
interaction between Sudan I and bovine serum albumin by spectro‐
scopic methods. Journal of Molecular Structure, 888, 152–159. https​://
doi.org/10.1016/j.molst​ruc.2007.11.043
Zhuang, J. X., Hu, Y. H., Yang, M. H., Liu, F. J., Qiu, L., Zhou, X. W., &
Chen, Q. X. (2010). Irreversible competitive inhibitory kinetics of car‐
dol triene on mushroom tyrosinase. Journal of Agricultural and Food
Chemistry, 58, 12993–12998. https​://doi.org/10.1021/jf103​723k
How to cite this article: Huang Q, Chai W‐M, Ma Z‐Y, et al.
Antityrosinase mechanism of ellagic acid in vitro and its
effect on mouse melanoma cells. J Food Biochem.
2019;00:e12996. https​://doi.org/10.1111/jfbc.12996​